TMA technology can be applied for the determination of diagnostic markers, as well as parameters related to the aggressiveness and other features of malignant tumors.
TMA is very useful for retrospective studies, i.e., for the investigation of thousands of molecular biological markers in thousands of histological specimens stored in pathology laboratories.
Similar to the DNA-chip, which allows the analysis of the genome mutations and abnormal gene expressions, the tissue micro-arrays enable the parallel processing of biological specimens. By using only one TMA, researchers have the opportunity for simultaneous comparative examination of various molecular parameters (DNA, RNA molecular biological techniques and immunohistochemistry/immunocytochemistry of antigens) in histological specimens of several malignant tumor tissues.
Numerous monoclonal antibodies, which determine the genome and phenotype of intact and pathological (mainly malignant) cells, and which are specific for novel antigen-determinants, are being developed and introduced for diagnostic and therapeutic applications (immunomorphology). Diagnostic measures based on genetic methods, which can identify certain segments of DNA and/or RNA, are increasingly used (molecular biology). Several laboratories are involved in evaluating their tissue reactivity and potential applicability in the most prevalent tumors.
As a consequence, this diagnostic technique indeed seems to be widely used.
In the diagnostic technique, histology specimens from different tumor tissues are embedded in properly arranged sets into paraffin for the preparation of TMA. The TMA block is cut giving the various tumor tissues as point-like items in the histological light microscopic sections. Since each TMA block may render hundreds of parallel, 34 micrometers thick serial sections, hundreds of molecular parameters can be examined on the same micro-array. Several tumor markers can be analyzed within weeks, while this would take months per tissue specimen using the traditional method.
According to our current knowledge, reactions made on standard-size histological sections (from frozen and/or fixed-embedded tissues) and cytological samples (smears, sediments, imprints, cytocentrifugates, etc) are useful for the identification of molecular markers of tumors and for the prediction of disease progression. At the same time, only a limited number of samples can be examined due to technical and financial reasons, the evaluation is time-consuming and comparative, and reproducible examinations among different laboratories are difficult to carry out. First of all, die lack of reproducibility is the disadvantage of the so-called “multi-tissue block” technique that contains parts from various tissues/blocks in a simple histological block. Moreover, the distribution of individual molecular markers within the malignant tissue is often quite heterogeneous and, thus, a minute tissue part does not properly represent the biological features of the entire tumor cell population.
Application of the technique reminiscent of or based on the “chip” technology, i.e. the tissue micro-arrays, enables the investigation of hundreds or thousands of tissue specimens.
The knowledge in the field of medical biotechnological basic research is being multiplied, and its employment confirms both diagnostic and therapeutic measures. Nevertheless, the related applied research, research and development (R&D), and clinical testing processes require years or even a decade and proportional financial means. In accordance with our invention, the TMA building manual set shortens the necessary testing phase in an accurate and cost-efficient way.
The information in U.S. Pat. No. 4,820,504 represents a well-known and widely used procedure, which serves for the preparation of a sample containing multiple tissue specimens. Its advantages include the simple procedure that can be applied in almost every laboratory equipped with routine and research facilities, low costs, and no need for procurement of new devices. However, the disadvantage of the technique is that the consecutive, side by side embedded tissue samples are randomly situated, not organized into lines and columns, which makes the evaluation rather difficult, and the automatic (image analyzing) processing almost impossible or complicated and slow. In addition, the size of the individual tissue samples are merely roughly similar to each other. A new instrument is not set forth in the description of die procedure.
Our procedure and our invention are aimed at addressing these disadvantages.
U.S. Pat. No. 5,002,377 discloses an instrument which organizes tissue samples into regular lines and columns, and so gives a multiple tissue block. Its patent holder is the same as that of the previous instrument who is a well-known expert in this field, although this technique has not become widely used. Its obvious advantage is that application of this instrument enables the preparation and embedding of regular, almost identical size tissue samples. Nevertheless, its disadvantage lies in the fact that the individual tissue columns may be, though only slightly, shifted during block preparation. The largest disadvantages of the instrument are its incompatibility with the most frequently applied and best known laboratory instruments (devices and semi-fixed assets) and, on the other hand, the need for three additional tools and the significant manual skills for their application. The first instrument includes multiple parallel cutting plates, which can cut out tissue columns from tissue samples, but which can be applied exclusively on larger specimens. The other two components make possible the embedding of cut tissue columns into a single paraffin block.
U.S. Pat. No. 6,103,518 discloses a table device giving useful tissue micro-arrays. The procedure is based on the well-oriented technique of the recipient places of the paraffin block and the sampling from the selected sites of donor blocks (tissue cores). Besides its aforementioned advantages, its high price, complicated mechanical settings, and the need for one-by-one preparation of recipient holes of the recipient block represent serious drawbacks in the daily practice.
PCT Publication No. WO 01/51910 presents a simple paraffin block construction procedure that can be used for the production of tissue micro-array series in which a mould that consists of several elements has been formed. On using the mould, a basic body is moulded that has regularly arranged openings into which the issue samples are then placed. Following this, the basic body containing the tissue samples is heated up to the softening temperature limit in the interest of the basic body being able to combine with the liquid paraffin poured onto one of the surfaces in the following phase of the procedure and so form a single unit with it. This liquid paraffin layer serves to assist in the fixing of the paraffin block holder to the paraffin block containing the cylindrical holes and the tissue samples contained in them.
However, a significant disadvantage of this solution is that the paraffin block that contains the cylinders with the tissue samples in them is subjected to a significant heat effect. Due to the heat effect and the re-softening of the paraffin, the tissue cylinders move, which can cause problems when making the sections. Another disadvantage of the subsequent heat effect is that the amount of heat required to soften the paraffin may damage the tissue sample cylinders, which may cause irreversible damage from the point of view of the immune staining, as this results in a change to the protein-based antigens, and this can lead to the sample being unusable.
Another significant disadvantage of the solution is that, in the paraffin block poured in two phases, due to the shear forces occurring when producing the section, the paraffin block may come apart at the border between the two layers made at two separate points in time, as a result of which the paraffin block may separate from the block holder, making it impossible to make the desired sections from the tissue cylinders.
Examples suitable for the production of paraffin blocks used for the production of tissue samples can also be found in U.S. Pat. No. 6,383,801 and, furthermore, in PCT Publication Nos. WO 01/98525, WO 99/44063, WO 01/220861 and WO 01/42796.